Antagonists and methods for inhibiting angiogenesis

ABSTRACT

The invention describes methods for inhibiting angiogenesis in a tissue by administering an antagonist that specifically binds to a proteolyzed or denatured collagen but not to native triple helical forms of the collagen. Antagonists of the invention can target, for example, denatured collagens type-I, type-II, type-III, type-IV, type-V and combinations thereof. Methods utilizing such antagonists for theraputic treatment of tumor growth, tumor metastasis or of restenosis also are described, as are methods to use such antagonists as diagnostic markers of angiogenesis is normal or diseased tissues both in vivo and ex vivo. Antagonists include monoclonal antibodies referred to as HUI77, HUIV26, and XL313.

RELATED APPLICATION DATA

This application is a divisional of Ser. No. 09/478,977, filed on Jan.6, 2000, entitled “Methods and Composition for Angiogenesis Inhibition,”which claims priority to provisional application Ser. No. 60/114,877filed Jan. 6, 1999, provisional application Ser. No. 60/114,878 filedJan. 6, 1999, provisional application Ser. No. 60/152,496 filed Sep. 2,1999, provisional application Ser. No. 60/143,534 filed Jul. 13, 1999.The entire disclosures of these applications are incorporated herein byreference.

FIELD OF INVENTION

The invention relates generally to the field of medicine, and relatesspecifically to methods and compositions for inhibiting angiogenesis ina tissue or detecting angiogenesis using antagonists of denatured orproteolyzed forms of collagen including, but not limited to types I, II,III, IV and V.

BACKGROUND

Tumor growth and metastasis impacts a large number of people each year.In fact, it is estimated that well over 600,000 new cases of cancer willbe diagnosed in the coming year in the United States alone (Vamer, J.A., Brooks, P. C., and Cheresh, D. A. (1995) Cell Adh. Commun. 3,367-374). Importantly, numerous studies have suggested that the growthof all solid tumors requires new blood vessel growth for continuedexpansion of the tumors beyond a minimal size (Varner et al. 1995;Blood, C. H. and Zetter, B. R. (1990) Biochim. Biophys. Acta.1032:89-118; Weidner, N. et al. (1992) J. Natl. Cancer Inst.84:1875-1,887; Weidner, N. et al. (1991). N. Engl. J. Med. 324:1-7;Brooks, P. C. et al. (1995) J. Clin. Invest. 96:1815-1822; Brooks, P. C.et al. (1994) Cell 79:1157-1164; Brooks, P. C. et al (1996). Cell 85,683-693; Brooks, P. C. et al. (1998) Cell 92:391-400. Significantly, awide variety of other human diseases also are characterized byunregulated blood vessel development, including ocular diseases such asmacular degeneration and diabetic retinopathy. In addition, numerousinflammatory diseases also are associated with uncontrolledneovascularization such as arthritis and psoriasis (Varner et al. 1995).Angiogenesis is the physiological process by which new blood vesselsdevelop from pre-existing vessels (Varner et al. 1995; Blood and Zetter1990; Weidner et al. 1992). This complex process requires cooperation ofa variety of molecules including growth factors, cell adhesionreceptors, matrix degrading enzymes and extracellular matrix components(Varner et al. 1995; Blood and Zetter 1990; Weidner et al. 1992). Thus,therapies designed to block angiogenesis may significantly effect thegrowth of solid tumors. In fact, clear evidence has been provided thatblocking tumor neovascularization can significantly inhibit tumor growthin various animal models, and human clinical data is beginning tosupport this contention as well (Varner, J. A., Brooks, P. C., andCheresh, D. A. (1995) Cell Adh. Commun. 3, 367-374). Importantly,numerous studies have suggested that the growth of all solid tumorsrequires new blood vessel growth for continued expansion of the tumorsbeyond a minimal size (Varner et al. 1995; Blood and Zetter 1990;Weidner et al. 1992; Weidner et al. 1991; Brooks et al. 1995; Brooks etal. 1994; Brooks et al. 1997).

To this end, many investigators have focused their anti-angiogenicapproaches towards growth factors and cytokines that initiateangiogenesis (Varner et al. 1995; Blood and Zener 1990; Weidner et al.1992; Weidner et al. 1991; Brooks et al. 1995; Brooks et al. 1994;Brooks et al. 1997). However, there is a large number of distinct growthfactors and cytokines which have the capacity to stimulate angiogenesis.The therapeutic benefit of blocking a single cytokine may have onlylimited benefit due to this redundancy. However, little attention hasbeen directed to other anti-angiogenic tar-gets. Recent studies havesuggested that angiogenesis requires proteolytic remodeling of theextracellular matrix (ECM) surrounding blood vessels in order to providea microenvironment conducive to new blood vessel development (Varner etal. 1995; Blood and Zetter 1990; Weidner et al. 1992; Weidner et al.1991; Brooks et al. 1995; Brooks et al. 1994; Brooks et al. 1997). Theextracellular matrix protein collagen makes up over 25% of the totalprotein mass in animals and the majority of protein within the ECM.Collagen is a fibrous multi-chain triple helical protein that exists innumerous forms (Olsen, B. R. (1995) Curr. Opin. Cell Biol. 7, 720-727;Van der Rest, M., and Garrone, R. (1991) FASEB 5, 2814-2823). At least18 genetically distinct types of collagen have been identified, many ofwhich have distinct tissue distributions and functions (Olsen 1995; Vander Rest and Garrone 1991). Collagen type-I is the most abundantcollagen type in the extracellular matrix. Collagen type-I, type-III,collagen type-IV and collagen type-V have been shown to be associatedwith all pre-existing blood vessels in vivo. Collagens type-I andtype-IV are composed of major chains designated α1(I) and α2(I) andα1(IV) and α2(IV) respectively. The mature collagen molecule is composedof two α1 chains and one α2 chain twisted into a triple helix. In vivo,collagen is normally found in the mature triple helical form.Denaturation of the native three dimensional structure of mature triplehelical collagen may expose cryptic regulatory regions that controlangiogenesis. Antagonism of these cryptic regulatory regions couldprovide an unrecognized means for the diagnosis and inhibition ofangiogenesis.

It has been proposed that inhibition of angiogenesis would be a usefultherapy for restricting tumor growth. Inhibition of angiogenesis hasbeen proposed by (1) inhibition of release of “angiogenic molecules”such as βFGF (fibroblast growth factor), (2) neutralization ofangiogenic molecules, such as by use of anti-βFGF antibodies, and (3)inhibition of endothelial cell response to angiogenic stimuli. Thislatter strategy has received attention, and Folkman et al., CancerBiology, 3:89-96 (1992), have described several endothelial cellresponse inhibitors, including collagenase inhibitors, basement membraneturnover inhibitors, angiostatic steroids, fungal-derived angiogenesisinhibitors, platelet factor 4, thrombospondin, arthritis drugs such asD-penicillamine and gold thiomalate, vitamin D₃ analogs,alpha-interferon, and the like that might be used to inhibitangiogenesis. For additional proposed inhibitors of angiogenesis, seeBlood and Zetter 1990; Moses et al. (1990) Science 248:1408-1410; Ingberet al. (1988) Lab. Invest., 59:44-51; and U.S. Pat. Nos. 5,092,885,5,112,946, 5,192,744, and 5,202,352. None of the inhibitors ofangiogenesis described in the foregoing references target denatured orproteolyzed collagens.

SUMMARY

The present invention provides antagonists of denatured or proteolyzedcollagens that can inhibit angiogenesis. Antagonists specifically bindto a denatured or proteolyzed collagen, but bind with substantiallyreduced affinity to native forms of the same collagen. Antagonists canbe specific for any denatured collagen, including denatured collagentype-I, denatured collagen type-II, denatured collagen type-III,denatured collagen type-IV or denatured collagen type-V, or forcombinations thereof. For example, in one embodiment, an antagonist isspecific for denatured collagen type-I relative to native triple helicalcollagen type-I but binds with substantially reduced affinity to otherdenatured collagens, such as collagen type-IV. In another embodiment, anantagonist is specific for denatured collagen type-IV. An antagonist canalso be specific for denatured collagen types I, II, III, IV and V.

An antagonist can be an antibody or functional fragment thereof, thatimmunoreacts with denatured collagen but immunoreacts to a substantiallylesser extent with the native form of the collagen. Antibodies can bemonoclonal or polyclonal. An antagonist also can be a polypeptide orpeptide with specificity for a denatured collagen, but not for a nativeform of the collagen. Antagonists also can be non-peptidic compoundssuch as a small organic molecule or an oligonucleotides.

The invention therefore describes methods for inhibiting angiogenesis ina tissue comprising administering to the tissue a composition comprisingan angiogenesis-inhibiting amount of an antagonist ofdenatured/proteolyzed collagen.

The tissue to be treated can be any tissue in which inhibition ofangiogenesis is desirable, such as diseased tissue whereneo-vascularization is occurring. Exemplary tissues include inflamedtissue, solid tumors, metastases, tissues undergoing restenosis, and thelike.

The invention also provides methods for detecting angiogenesis in atissue by contacting an antagonist of the invention with the tissue.Such methods are appropriate for use both ex vivo and in vivo.

Methods also are provided for detecting tumorous tissue, metastases, andtumor invasion into a tissue by contacting an antagonist of theinvention with a tissue either ex vivo or in vivo.

The invention also provides methods for screening antagonists that bindspecifically to a denatured collagen or collagens, but bind withsubstantially reduced affinity to the native form of the collagen orcollagens and can inhibit angiogenesis.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates Mab HUI77 reactivity with extracellular matrixcomponents in solid phase ELISA. Microtiter plates (96 well) were coatedwith extracellular matrix components including native collagen type-Iand type-IV, denatured collagen type-I and type-IV, vitronectin,fibronectin and fibrinogen, each at a concentration of 10 micrograms(ug) per milliliter (mL). Wells of the microtiter plate were blockedwith 1% BSA in PBS for 1 hour at 37° C. Mab HUI77 was added to the wellsat a concentration of 1 ug/mL and allowed to incubate for 2 hours at 37°C. After incubation, immunoreactivity was detected by incubation withgoat anti-mouse peroxidase labeled secondary antibody. Immunoreactivitywas measured with an ELISA plate reader at 490 nm usingo-phenylenediamine as a substrate. Coll-I (native triple helicalcollagen type-I). Denatured Coll-I (Denatured Collagen type-I). Coll-IV(native triple helical collagen type-IV). Denatured Coll-IV (denaturedcollagen type-IV). VN (Vitronectin). FN (Fibronectin). FB (fibrinogen).Data bars represent the mean Optical Density (O.D) ± standard deviationsfrom triplicate wells.

FIG. 2 demonstrates Mab HUI77 reactivity with genetically distinct formsof collagen. Microtiter plates were coated with distinct forms ofcollagen at a concentration of 10 μg/mL. Wells of the microtiter plateswere blocked with 1% BSA in PBS for 1 hour at 37° C. Immunoreactivitywas detected by incubation with goat anti-mouse peroxidase labeledsecondary antibody. Immunoreactivity was measured by determining theoptical density with an ELISA plate reader at 490 nm. Coll-I; triplehelical Collagen-I. Den Coll-I; denatured Collagen-I. Coll-II; triplehelical Collagen-II. Den Coll-II; denatured Collagen-II. Coll-III;triple helical Collagen-III. Den Coll-III; denatured Collagen-III.Coll-IV; triple helical Collagen-IV. Den Coll-IV; denatured Collagen-IV.Coll-V; triple helical Collagen-V. Den Coll-V; denatured Collagen-V.Data bars represent the mean O.D. ± standard deviation from triplicatewells.

FIG. 3 shows that Mab HUI77 identifies denatured collagen surroundinghuman melanoma tumors in vivo. The generation and localization ofdenatured collagen in vivo was studied by indirect immunofluoresence onfrozen tissue sections of human melanomas from both the human mousechimeric model and human tumor biopsies. Frozen tissue sections fromhuman melanomas were fixed in acetone, blocked with 1% BSA andco-stained with Mab HUI77 and polyclonal antibody directed to α_(v)integrin expressed on the human melanoma cells. Antibody binding wasdetected by incubation with goat-anti-mouse FITC conjugated and goatanti-rabbit rhodamine conjugated secondary antibody. Left panelsindicate human melanoma tumor biopsy (630X). Right panels indicate M21human melanoma cell line grown within full thickness human skin (200X).Red indicates α_(v) integrin expression and green indicates denaturedcollagen expression. Yellow indicates co-localization of α_(v) integrinsand denatured collagen.

FIG. 4 shows that Mab HUI77 identifies denatured collagen surroundinghuman melanoma tumor associated blood vessels. The generation andlocalization of denatured collagen in vivo was studied by indirectimmunofluoresence on frozen tissue sections of human melanoma tumorbiopsies. Frozen tissue sections from human melanomas were fixed inacetone, blocked with 1% BSA and co-stained with Mab HUI77 andpolyclonal antibody directed to factor VIII, a known marker of bloodvessels. Antibody binding was detected by incubation withgoat-anti-mouse fluorescein isothiocyanate (FITC) conjugated and goatanti-rabbit rhodarnine conjugated secondary antibodies. Left panel showshuman melanoma tumor biopsy staining for factor VIII (Red) outlining atumor blood vessel. Right panel shows denatured collagen (Green)surrounding the human tumor associated blood vessel.

FIG. 5 demonstrates the effects of Mab HUI77 on human endothelial celladhesion. Microtiter plates (96 wells) were coated with either native ordenatured collagen type-I or type-IV at a concentration of 10 μg/mL.Wells of the microtiter plate were blocked with 1% BSA in PBS for 1 hourat 37° C. Human endothelial cells (HUVECs) were then allowed to attachto the coated wells in the presence or absence of purified Mab HUI77 (50μg/mL) or an isotype matched control antibody at a concentration of 50ug/mL for 30 minutes. The non-attached cells were removed by washing andthe attached cells were stained with crystal violet. Cell adhesion wasquantified by measuring the optical density (O.D.) of eluted dye at 600nm. Data Bars represent the mean O.D ± standard deviation fromtriplicate wells. Data are represented as percent of control.

FIG. 6 demonstrates the effects of Mab HUI77 on human endothelial cellmigration. Membranes from transwell migration chambers were coated witheither denatured collagen type-I or type-IV at a concentration of 25μg/mL. Human endothelial cells (HUVECs) were allowed to migrate in thepresence or absence of purified Mab HUI77 or an isotype matched controlantibody (100 ug/mL) for a total of 6 hours. Cells remaining on the topside of the membrane were removed and the cells which had migrated tothe underside of the membrane were stained with crystal violet. Cellmigration was quantified by measuring the optical density (O.D) ofeluted dye at 600 nm with a microplate reader. Data Bars represent themean O.D ± standard deviation from triplicate wells. Data are expressedas percent of control.

FIG. 7 demonstrates the effects of systemic administration of purifiedMab HUI77 on angiogenesis in vivo. Filter discs saturated with βFGF wereplaced on the Chorioallantoic membranes (CAMs) of 10 day old chickembryos. Twenty four hours later the embryos received a singleintravenous injection with 20 ug of Mab HUI77 or a control. At the endof a 3 day incubation period the filter discs and surrounding CAMtissues were removed and angiogenesis was quantified by counting thenumber of blood vessel branch points within the area of the filter disc.Examples of CAM tissue from a typical experiment are shown.

FIG. 8 shows the quantification of the angiogenesis experiments with MabHUI77. Data bars represent the mean ± standard errors of 5 to 10 embryosper condition. Angiogenesis index is equal to the number of branchpoints from experimentally treated embryos minus the number of branchpoints form CAMs in the absence of βFGF.

FIG. 9 shows the effects of systemic administration of purified MabHUI77 on tumor-growth in vivo. A). CS-1 melanoma tumor cells (5×10⁶)were inoculated on the CAMs of 10 day old chick embryos. Twenty fourhours later the embryos received a single intravenous injection of 100ug of purified Mab HUI77 or control. The embryos were allowed toincubate for a total of 7 days. At the end of the 7 day incubationperiod, the resulting tumors were resected and wet weights determined.Photographs show representative tumors taken from embryos treated withor without Mab HUI77.

FIG. 10 shows the quantification of the wet weight of the tumors. Databars represent the mean ± the standard errors of the tumor weights from5 to 12 embryos per condition.

FIG. 11 demonstrates Mab HUIV26 reactivity with extacellular matrixcomponents in solid phase ELISA. Microtiter plates were coated withextracellular matrix components, each at a concentration of 25 ug/ml.A). Mab HUIV26 was added at a concentration of 1 ug/ml, followed 1 hourlater with goat anti-mouse peroxidase labeled IgG. Denatured collagentype-I and collagen type-IV were prepared by boiling for 15 minutesbefore coating the plates. All data were corrected for any non-specificbinding of secondary antibody. Data bars represent the mean opticaldensity (O.D.) ± standard deviations from triplicate wells. B).Microtiter wells were coated with triple helical collagen type-IV at 25ug/ml. Concentrated (20×) HUVEC conditioned media was added to the wellsin the presence or absence of EDTA, Aprotinin or both and allowed toincubate for 1, 6 and 24 hours. The plates were next washed, blocked andincubated with Mab HUIV26 or control antibody. All data were correctedfor non-specific secondary antibody binding. Data bars represent themean optical density (O.D.) ± standard deviations from triplicate wells.The following abbreviations are used in the figure Coll-I, collagentype-I; Coll-IV, collagen type-IV.

FIG. 12 demonstrates that Mab HUIV26 identifies denatured collagen-IVSurrounding Angiogenic Blood Vessels Within the Chick ChorioallantoicMembrane (CAM). The generation and localization of denatured collagen-IVin vivo was studied by indirect immunofluoresence on frozen tissuesections of CAM tissue from both bFGF induced and tumor inducedangiogenesis. Frozen CAM tissue sections were fixed in acetone, blockedwith 1% BSA and co-stained with Mab HUIV26 and polyclonal antibodiesdirected to either the collagen-IV degrading enzyme MMP-2 or FactorVIII. Antibody binding was detected by incubation with goat-anti-mouseFITC conjugated and goat anti-rabbit rhodamine conjugated secondaryantibody. Top panels show co-localization of denatured collagen-IV andMMP-2 surrounding angiogenic blood vessels. Bottom Panels showco-localization of denatured collagen-IV and factor VIII surroundingangiogenic blood vessels. Left panels indicate CAM tissue stimulatedwith bFGF. Right panels indicate CAM tissue with CS1 melanoma tumorsgrowing within it. Red color in the top panels indicate MMP-2 expressionand in the bottom panels it indicates factor VIII expression. Greencolor in both the top and bottom panels indicate denatured collagen-IVexpression. Yellow indicates co-localization.

FIG. 13 shows that Mab HUIV26 identifies denatured collagen type-IVsurrounding human melanoma tumor associated blood vessels. Thegeneration and localization of denatured collagen-IV in vivo was studiedby indirect immunofluoresence on frozen tissue sections of humanmelanomas tumor biopsies. Frozen tissue sections from human melanomaswere fixed in acetone, blocked with 1% BSA and co-stained with MabHUIV26 and polyclonal antibody directed to factor VIII a known marker ofblood vessels. Antibody binding was detected by incubation withgoat-anti-mouse FITC conjugated and goat anti-rabbit rhodamineconjugated secondary antibody. Red indicates factor VIII expression andmarks the human blood vessels. Green indicates denatured collagen-IVspecifically associated with the tumor associated angiogenic bloodvessels.

FIG. 14 demonstrates the effects of Mab HUIV26 on human endothelial celladhesion. Microtiter plates (96 wells) were coated with either native ordenatured collagen type-IV. Wells of the microtiter plate were nextblocked with 1% BSA in PBS for 1 hour at 37° C. Human endothelial cellswere then allowed to attach to the coated wells in the presence orabsence of purified Mab HUIV26 or an isotype matched control antibody ata concentration of 100 ug/ml for 30 minutes. The non-attached cells wereremoved by washing and the attached cells were stained with crystalviolet. Cells were next incubated with 10% acetic acid and cell adhesionwas quantified by measuring the optical density (O.D) of eluted dye at600 nm. Data Bars represent the mean O.D ± standard deviation fromtriplicate wells.

FIG. 15 shows the effects of Mab HUIV26 on human endothelial cellmigration. Membranes from transwell migration chambers were coated witheither native or denatured collagen type-IV. Human endothelial cellswere then allowed so migrate to the underside of the coated membranes inthe presence or absence of purified Mab HUIV26 or an isotype matchedcontrol antibody (100 ug/ml ) for a total of 6 hours. Cells remaining onthe top side of the membrane were removed and the cells which hadmigrated to the underside of the membrane were stained with crystalviolet. Membranes were next incubated with 10% acetic acid and cellmigration was quantified by measuring the optical density (O.D) ofeluted dye at 600 nm. Data Bars represent the mean O.D ± standarddeviation from triplicate wells.

FIG. 16 demonstrates the effects of systemic administration of purifiedMab HUIV26 on angiogenesis in vivo. Filter discs saturated with bFGFwere place on the Chorioallantoic Membranes (CAMs) of 10 day old chickembryos. Twenty four hours latter the embryos received a singleintravenous injection with 20 ug of Mab HUIV26 or a control. At the endof a 3 day incubation period the filter discs and surrounding CAMtissues were removed and angiogenesis was quantified by counting thenumber of blood vessel branch points with the area of the filter disc.FIG. 6 shows examples of CAM tissue from a typical experiment.

FIG. 17 shows quantification of the angiogenesis-experiments with MabHUIV26. Data bars represent the mean ± standard errors of 5 to 10embryos per condition. Angiogenesis index is equal to the number ofbranch points from experimental treated embryos minus the number ofbranch points form CAMs in the absence of bFGF.

FIG. 18 shows the effects of systemic administration of purified MabHUIV26 on tumor growth in vivo. CS-1 melanoma tumor cells (5×10⁶) wereinoculated on the CAMs of 10 day old chick embryos. Twenty four hourslatter the embryos received a single intravenous injection of 20 μg ofpurified Mab HUIV26 or control. The embryos were allowed to incubate fora total of 7 days. Photographs represent examples of tumors from controlor HUIV26 treated embryos within the CAM tissue.

FIG. 19 shows size comparison of resected tumors. At the end of the 7day incubation period the resulting tumors were resected and analyzedfor overall size and wet weight. Photograph represents a comparison ofthe size of resected tumors from control or Mab HUIV26 treated embryos.

FIG. 20 shows quantification of the wet weight of the tumors. Data barsrepresent the mean ± the standard errors of the tumor weight from 5 to10 embryos per condition.

FIG. 21 shows the effects of Mab HUIV26 on tumor-growth assessed in aSCID mouse tumor model system. SCID mice were injected subcutaneouslywith 2×10⁶ M21 human melanoma cells. Three days later a 24 day treatmentwas initiated with daily intraperitoneal injections of either 100 ug ofeither Mab HUIV26, an isotype matched control antibody, or withouttreatment. Tumor volume was monitored by caliper measurements and tumorvolume was determined. Five mice were included in each group. The datarepresent the mean ± standard errors of the tumor volumes for eachexperimental condition. Similar results were obtained in two independentexperiments where five or ten mice were included in each experimentalcondition.

FIG. 22 shows an analysis of Mab HUIV26 reactivity with RGD containingcollagen peptides. Microtiter plates were coated with 50 ul of eitherRGD containing collagen peptides (100 ug/ml) (A) or denatured humancollagen type-IV (B). Plates were blocked with 1% BSA in PBS to preventnon-specific binding. Mab HUIV26 (1.0 ug/ml, 50 ul/well) was allowed tobind to the coated plate for 1 hour at 37° C. Mab HUIV26 binding wasdetected by incubation with peroxidase labeled secondary antibody.Immunoreactivity was quantified by measuring optical density (O.D.) witha microtiter plate reader. A). Immunoreactivity of purified Mab HUIV26to RGD containing collagen peptides. B). Immunoreactivity of HUIV26binding to immobilized denatured collagen type-IV in the presence orabsence of soluble RGD containing collagen peptides or denaturedcollagen type-IV. Data bars represent the mean O.D. ± standard deviationfrom triplicate wells.

FIG. 23 shows human endothelial cord formation on denatured Collagentype-I. Flexible Millipore membranes were coated with either native ordenatured human collagen type-I. Human endothelial cells (HUVECs) wereallowed to interact with the collagen for 5 hours in the absence ofadded growth factors or serum.

FIG. 24 shows analysis of endothelial cell survival on collagen type-I.Microtiter wells were coated with either native or proteolyzed/denaturedhuman collagen type-I. HUVECs were next allowed to attach to the wellsin the absence of added growth factors or serum for 5 hours. At the endof the 5 hour incubation, attached cells were removed, fixed and stainedfor apoptosis with ApopTag kit and analyzed by flow cytometry. Dataindicated the mean ± standard deviations of the percentage of humanendothelial cells that were beginning to undergo apoptosis. Data werederived from triplicate wells.

FIG. 25 shows effects of cryptic collagen type-I domains on endothelialcord formation. Analysis of human collagen-I amino acid sequence andthree dimensional structure revealed potential cryptic domains thatwould be inaccessible within mature triple helical collagen-I. Syntheticpeptides were generated corresponding to 5 of these potential crypticdomains of human collagen-I. These cryptic domains were immobilized onmicrotiter wells and endothelial cord formation assays were conducted asdescribed above. As shown, all 5 collagen peptides supported endothelialcell adhesion by 1 hour following plating. However, human collagenpeptide-2 was shown to facilitate endothelial cord formation andpotentiate endothelial cell survival after 18 hours, while the otherpeptides showed little if any activity at the 18 hour time point.

FIG. 26 shows that cryptic domains of collagen type-I supportendothelial cell adhesion by distinct integrin receptors. Peptidesrepresenting cryptic domains of human collagen-I were immobilized onmicrotiter wells. Endothelial cells were allowed to attach to thepeptides in the presence or absence of function blocking antibodiesdirected to specific integrins. All peptides supported cell adhesion tovarying levels. Cell adhesion to all 5 peptides were dependent onligation of integrin αvβ3 since Mab LM609 directed to αvβ3 blocked celladhesion. Surprisingly, cell adhesion to peptide-2 was also dependent ona β1 integrin, since cell adhesion to this peptide was also blocked byP4C10 directed to β1 integrins.

FIG. 27 demonstrates the generation of Mabs reactive with crypticdomains of collagen type-I. Cryptic domains of collagen type-I were usedto generate Mabs. One of these Mabs termed XL313 was used for furtherstudy. Human collagen-I peptides were immobilized on microtiter wellsand purified Mab XL313 binding was assessed. As shown below, Mab XL313specifically recognized human collagen peptide-2. In addition Mab XL313also recognized collagen peptide-4, however, collagen peptide-4 is notpresent in mature collagen-I. XL313 did not react with other similarcollagen type-I peptides.

FIG. 28 shows that Mab XL313 specifically recognizes denatured humancollagen type-I. Microtiter wells were coated with either native ordenatured human collagen type-I or type-IV. The capacity of Mab XL313 tobind to native or denatured human collagen type-I and denatured collagentype-IV was assessed by solid phase ELISA. Mab XL313 specificallyrecognized denatured human collagen type-I but not native collagentype-I. In addition, Mab XL313 failed to bind to native or denaturedhuman collagen type-IV.

FIG. 29 shows that Mab XL313 inhibits angiogenesis in the chick.Angiogenesis was induced on the CAMs of 10 day old chick embryos withbFGF. Twenty-four hours later the embryos received a single IV injectionwith 50 μg of Mab XL313 or an isotope matched control. Three days later,angiogenesis was quantified by counting the number of blood vesselbranch points within the area of the filter disc. A; Representativeexamples of CAM tissue from a typical experiment.

FIG. 30 shows quantification of the angiogenesis experiments of FIG. 29.Data bars represent the mean ± standard errors of 5 to 10 embryos percondition.

FIG. 31 shows the effects of systemic administration of Mab XL313 onhuman fibrosarcoma tumor growth. HT1080 human fibrosarcoma cells (5×10⁶)were inoculated on the CAMs of 10 day old chick embryos. Twenty-fourhours later the embryos received a single intravenous injection ofpurified Mab XL313 at concentrations of 50 μg per embryo. After 7 days,tumors re resected and wet weights determined. Quantification of tumorsweight. Data bars represent the mean ± the standard errors from 5 to 10embryos per condition.

FIG. 32 shows that mutation in the MMP cleavage site of collagen-Iinhibits melanoma tumor growth in vivo. Transgenic mice that harbor amutation in the MMP cleavage site within collagen-I were used todetermine if proteolysis of collagen type-I may play a role in tumorgrowth and angiogenesis. Col a1 transgenic mice have a mutation whichinhibits binding of MMPs and cleavage of collagen type-I. Col a1 B6transgenic mice or wild type control B6 mice were injectedsubcutaneously with B16 transgenic melanoma tumor cells. Tumors wereallowed to develop for 11 days and tumor size was monitored withcalipers. As show below, B16 melanoma cells formed large proliferatingtumors within mice that can readily proteolyze collagen type-I. Incontrast, B16 melanoma cells exhibited little if any capacity to formtumors in the B6 Col a1 transgenic mice in which collagen type-Iproteolysis is inhibited. Data represent the mean ± standard errors ofthe tumor volumes from 5 mice per condition.

FIG. 33 shows that Mab XL313 inhibits tumor growth in B6 mice. Thegrowth of Lewis lung carcinoma tumors were examined in either wild typeB6 mice or Col a1 transgenic mice. A; Lewis lung carcinoma cells wereinjected subcutaneously into either wild-type B6 mice or B6 col a1transgenic mice. B; Comparison of Lewis lung carcinoma tumor growthwithin wild-type B6 or col a1 B6 transgenic mice; C; Wild-type B6control mice were injected with Lewis lung carcinoma cells. Twenty-fourhours later the mice were treated systematically with either Mab XL313or an isotope matched control antibody (100 μg/per injection). Tumorswere allowed to develop for 11 days and tumor sizes was monitored withcalipers. Data represent the mean ± standard errors of the tumor volumesfrom 5 mice per condition.

DESCRIPTION OF THE INVENTION

Collagens

The methods of the invention are suitable for use with a number ofcollagen molecules, including those from any animal. In one embodimentcollagens are human collagens. Collagens may also be from any mammalsuch as rat, mouse, pig, rabbit etc. or from a bird such as chicken.Generally, a collagen is an extracellular matrix protein containing a[Gly-Xaa-Xaa]_(n) sequence. Collagen types are well known in the art(see, e.g., Olsen, B. R. (1995) Curr. Op. Cell. Biol. 5:720-727;Kucharz, E. J. The Collagens: Biochemistry and Pathophysioloy.Springer-Verlag, Berlin, 1992; Kunn, K. in Structure and Function ofCollagen Types, eds. R. Mayne and R. E. Burgeson, Academic Press,Orlando). Human collagens are preferred collagens. Denatured collagenrefers to collagen that has been treated such that it no longerpredominantly assumes the native triple helical form. Denaturation canbe accomplished by beating the collagen. In one embodiment, collagen isdenatured by heating for about 15 minutes to about 100° C. Denaturationcan also be accomplished by treating the collagen with a chaotropicagent. Suitable chaotropic agents include, for example, guanidiniumsalts. Denaturation of a collagen can be monitored, for example, byspectroscopic changes in optical properties such as absorbance, circulardichroism or fluorescence of the protein, by nuclear magnetic resonance,by Raman spectroscopy, or by any other suitable technique. Denaturedcollagen refers to denatured full length collagens as well as tofragments of collagen. A fragment of collagen can be any collagensequence shorter than a native collagen sequences. For fragments ofcollagen with substantial native structure, denaturation can be effectedas for a native full-length collagen. Fragments also can be of a sizesuch that they do not possess significant native structure or possessregions without significant native structure of the native triplehelical form. Such fragments are denatured all or in part withoutrequiring the use of heat or of a chaotropic agent. The term denaturedcollagen encompasses proteolyzed collagen. Proteolyzed collagen refersto a collagen that has been fragmented through the action of aproteolytic enzyme. In particular, proteolyzed collagen can be preparedby treating the collagen with a metalloproteinase, such as MMP-1, MMP-2or MMP-9, or by treating the collagen with a cellular extract containingcollagen degrading activity or is that which occurs naturally at sitesof neovascularization in a tissue.

A cryptic epitope within a collagen is a sequence that is not exposedfor recognition within a native collagen, but is capable of beingrecognized by an antagonist of a denatured collagen. The sequence ofcryptic epitopes can be identified by determining the specificity of anantagonist. Candidate cryptic epitopes also can be identified, forexample, by examining the three dimensional structure of a native triplehelical collagen. Peptide sequences that are not solvent exposed or areonly partially solvent exposed in the native structure are potentialcryptic epitopes.

An epitope is that amino acid sequence or sequences that are recognizedby an antagonist of the invention. An epitope can be a linear peptidesequence or can be composed of noncontiguous amino acid sequences. Anantagonist can recognize one or more sequences, therefore an epitope candefine more than one distinct amino acid sequence target. The epitopesrecognized by an antagonist can be determined by peptide mapping andsequence analysis techniques well known to one of skill in the art.

Antagonists

Antagonists of the invention bind to a denatured collagen but bind withsubstantially reduced affinity to the native form of the collagen. A“substantially reduced affinity” is an affinity of about 3-fold lowerthan that for the denatured collagen, more preferably, about 5-foldlower, and even more preferably about 10-fold lower, and even morepreferably greater than 10-fold lower. Likewise, “substantially less”indicates a difference of at least about a 3 fold difference whenreferring to relative affinities. Antagonists are preferably specificfor any one of the denatured collagens types-I, II, III, IV or V andcombinations thereof. In one embodiment an antagonist binds to denaturedcollagen type-I but binds with substantially reduced affinity to nativecollagen type-I and to denatured collagens types II, III, IV and V. Inanother embodiment, an antagonist binds to denatured collagen type-IVbut binds with substantially reduced affinity to native collagentype-IV. In another embodiment an antagonist binds to denaturedcollagens type-I, type-II, type-III, type-IV and type-V but binds withsubstantially reduced affinity to native collagens type-I, type-II,type-III, type-IV and type-V.

Apparent affinities can be determined by methods such as an enzymelinked immunosorbent assay (ELISA) or any other technique familiar toone of skill in the art. True affinities can be measured by techniquesknown to one of skill in the art.

In one embodiment, peptides containing epitopes recognized by anantagonist can be used themselves. In one embodiment, epitopes definedby the monoclonal antibodies HUI77, HUIV26 and XL313 are themselves usedas antiangiogenic compositions.

Binding Assays

The invention also provides assay methods for identifying candidatedenatured collagen antagonists for use according to the present methods.In these assay methods candidate antagonists are evaluated for theirability to bind both denatured collagen and native collagen, andfurthermore can be evaluated for their potency in inhibitingangiogenesis in a tissue.

ELISA

The first assay measures binding of antagonists to denatured or nativecollagens in the solid phase by ELISA. The assay is useful with avariety of types of collagens, for example, the assay can be used withcollagens types, I, II, III, IV and V as well as for other extracellularmatrix components.

The assay also can be used to identify compounds which exhibitspecificity for denatured but not native forms of collagen. Thespecificity assay is conducted by running parallel ELISAs where apotential antagonist is screened concurrently in separate assay chambersfor the ability to bind denatured and native collagens.

Antagonists of denatured collagen can also be identified by theirability to compete for binding with an antagonist of the invention. Forexample, putative antagonists can be screened by monitoring their effecton the affinity of a known antagonist, such as HUI77, HUIV26 or XL313,in a binding assay, such as ELISA. Such antagonists likely have the samespecificity as HUI77, and recognize the same cryptic epitope. Putativeantagonists selected by such a screening method can bind either to thecollagen or to the antagonist. Antagonists can be selected from theputative antagonists by conventional binding assays to determine thosethat bind to the denatured collagen epitope but not to the knownantagonist.

Antagonists can also be identified by their ability to bind to a solidmatrix containing a denatured collagen. Such putative antagonists arecollected after altering solution conditions, such as saltconcentration, pH, temperature, etc. The putative antagonists arefurther identified by their ability to pass through, under appropriatesolution conditions, a solid matrix to which a native collagen has beenaffixed.

Angiogenesis Assays

Antagonists of the invention also can be assayed for their ability tomodulate angiogenesis in a tissue. Any suitable assay known to one ofskill in the art can be used to monitor such effects. Several suchtechniques are described herein.

The second assay measures angiogenesis in the chick chorioallantoicmembrane (CAM) and is referred to as the CAM assay. The CAM assay hasbeen described in detail by others, and further has been used to measureboth angiogenesis and neovascularization of tumor tissues. See Ausprunket al., Am. J. Pathol., 79:597-618 (1975) and Ossonski et al., CancerRes., 40:2300-2309 (1980).

The CAM assay is a well recognized assay model for in vivo angiogenesisbecause neovascularization of whole tissue is occurring, and actualchick embryo blood vessels are growing into the CAM or into the tissuegrown on the CAM.

As demonstrated herein, the CAM assay illustrates inhibition ofneovascularization based on both the amount and extent of new vesselgrowth. Furthermore, it is easy to monitor the growth of any tissuetransplanted upon the CAM, such as a tumor tissue. Finally, the assay isparticularly useful because there is an internal control for toxicity inthe assay system. The chick embryo is exposed to any test reagent, andtherefore the health of the embryo is an indication of toxicity.

A third assay measures angiogenesis is the in vivo rabbit eye model andis referred to as the rabbit eye assay. The rabbit eye assay has beendescribed in detail by others, and further has been used to measure bothangiogenesis and neovascularization in the presence of angiogenicinhibitors such as thalidomide. See D'Amato et al. (1994) Proc. Natl.Acad. Sci. 91:4082-4085.

The rabbit eye assay is a well recognized assay model for in vivoangiogenesis because the neovascularization process, exemplified byrabbit blood vessels growing from the rim of the cornea into the cornea,is easily visualized through the naturally transparent cornea of theeye. Additionally, both the extent and the amount of stimulation orinhibition of neovascularization or regression of neovascularization caneasily be monitored over time.

Finally, the rabbit is exposed to any test reagent, and therefore thehealth of the rabbit is an indication of toxicity of the test reagent.

A fourth assay measures angiogenesis in the chimeric mouse:human mousemodel and is referred to as the chimeric mouse assay. The assay has beendescribed in detail by others, and further has been described herein tomeasure angiogenesis, neovascularization, and regression of tumortissues. See Yan, et al. (1993) J. Clin. Invest. 91:986-996.

The chimeric mouse assay is a useful assay model for in vivoangiogenesis because the transplanted skin grafts closely resemblenonnal human skin histologically and neovascularization of whole tissueis occurring wherein actual human blood vessels are growing from thegrafted human skin into the human tumor tissue on the surface of thegrafted human skin. The origin of the neovascularization into the humangraft can be demonstrated by immunohistochemical staining of theneovasculature with human-specific endothelial cell markers.

The chimeric mouse assay demonstrates regression of neovascularizationbased on both the amount and extent of regression of new vessel growth.Furthermore, it is easy to monitor effects on the growth of any tissuetransplanted upon the grafted skin, such as a tumor tissue. Finally, theassay is useful because there is an internal control for toxicity in theassay system. The chimeric mouse is exposed to any test reagent, andtherefore the health of the mouse is an indication of toxicity.

Antibodies

The present invention describes, in one embodiment, denatured collagenantagonists in the form of antibodies which bind to denatured collagenbut bind to native collagen with a substantially reduced affinity.Antibody antagonists also can inhibit angiogenesis. The invention alsodescribes cell lines which produce the antibodies, methods for producingthe cell lines, and methods for producing the monoclonal antibodies.

Antibodies of the invention can be monoclonal or polyclonal. In oneembodiment, antibodies used are monoclonal. A monoclonal antibody ofthis invention comprises antibody molecules that immunoreact withisolated denatured collagen, but immunoreact with a substantiallyreduced affinity with the native form of the collagen. In oneembodiment, an antibody of the invention recognizes denatured collagentype-I with an affinity at least about 3-fold, more preferably at leastabout 5-fold and most preferably at least about 10-fold higher than thatfor denatured collagen type-I. An antibody of the invention also canbind to preferably to denatured collagen type-IV and binds withsubstantially reduced affinity to native collagen type-IV. Antibodies ofthe invention also can bind to each of collagens types I, II, III, IVand V and bind to the native forms of each collagen with substantiallyreduced affinity.

Preferred monoclonal antibodies which preferentially bind to denaturedcollagen include monoclonal antibodies having the immunoreactioncharacteristics of mAb HUI77, mAb HUIV26 or mAb XL313.

Antibodies antagonists of the invention can be generated according to anumber of methods known to one of skill in the art. For example, ananimal can be immunized with a denatured collagen or fragment thereof.Antibodies thus generated can be selected both for their ability to bindto denatured proteolyzed collagen and for a substantially reducedaffinity for the native form of the same collagen. Antibodies can, forexample, be generated by the method of “subtractive immunization” (see,e.g., Brooks, P. C. et al. (1993) J. Cell. Biol. 122:1351-1359.)

The term “antibody or antibody molecule” in the various grammaticalforms is used herein as a collective noun that refers to a population ofimmunoglobulin molecules and/or immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain an antibodycombining site or paratope.

An “antibody combining site” is that structural portion of an antibodymolecule comprised of heavy and light chain variable and hypervariableregions that specifically binds antigen.

Exemplary antibodies for use in the present invention are intactimmunoglobulin molecules, substantially intact immunoglobulin moleculesand those portions of an immunoglobulin molecule that contain theparatope, including those portions known in the art as Fab, Fab′,F(ab′)₂ and F(v), and also referred to as antibody fragments.

In another preferred embodiment, the invention contemplates a truncatedimmunoglobulin molecule comprising a Fab fragment derived from amonoclonal antibody of this invention. The Fab fragment, lacking Fcreceptor, is soluble, and affords therapeutic advantages in serum halflife, and diagnostic advantages in modes of using the soluble Fabfragment. The preparation of a soluble Fab fragment is generally knownin the immunological arts and can be accomplished by a variety ofmethods.

For example, Fab and F(ab′)₂ portions (fragments) of antibodies areprepared by the proteolytic reaction of papain and pepsin, respectively,on substantially intact antibodies by methods that are well known. Seefor example, U.S. Pat. No. 4,342,566 to Theofilopolous and Dixon. Fab′antibody portions also are well known and are produced from F(ab′).sub.2 portions followed by reduction of the disulfide bonds linking thetwo heavy chain portions as with mercaptoethanol, and followed byalkylation of the resulting protein mercaptan with a reagent such asiodoacetamide. An antibody containing intact immunoglobulin moleculesare preferred, and are utilized as illustrative herein.

The phrase “monoclonal antibody” in its various grammatical forms refersto a population of antibody molecules that contain only one species ofantibody combining site capable of immunoreacting with a particularepitope. A monoclonal antibody may therefore contain an antibodymolecule having a plurality of antibody combining sites, eachimmunospecific for a different epitope, e.g., a bispecific monoclonalantibody.

A monoclonal antibody is typically composed of antibodies produced byclones of a single cell called a hybridoma that secretes (produces) onlyone kind of antibody molecule. The hybridoma cell is formed by fusing anantibody-producing cell and a myeloma or other self-perpetuating cellline. The preparation of such antibodies was first described by Kohlerand Milstein, Nature 256:495-497 (1975), which description isincorporated by reference. Additional methods are described by Zola,Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc. (1987).The hybridoma supernatant so prepared can be screened for the presenceof antibody molecules that immunoreact with denatured collagens.

Briefly, to form the hybridoma from which the monoclonal antibodycomposition is produced, a myeloma or other self-perpetuating cell lineis fused with lymphocytes obtained from the spleen of a mammalhyperimmunized with a source of denatured collagen.

It is preferred that the myeloma cell line used to prepare a hybridomabe from the same species as the lymphocytes. Typically, a mouse of thestrain 129 G1 X.sup.+ is the preferred mammal. Suitable mouse myelomasfor use in the present invention include thebypoxanthine-aminopterin-thymidine-sensitive (HAT) cell linesP3X63-Ag8.653, and Sp2/0-Ag14 that are available from the American TypeCulture Collection, Rockville, Md., under the designations CRL 1580 andCRL 1581,respectively.

Splenocytes are typically fused with myeloma cells using polyethyleneglycol (PEG) 1500. Fused hybrids are selected by their sensitivity to aselective growth medium, such as HAT (hypoxanthine aminopterinthymidine) medium. Hybridomas producing a monoclonal antibody of thisinvention are identified using the enzyme linked immunosorbent assay(ELISA) described in the Examples.

A monoclonal antibody of the present invention also can be produced byinitiating a monoclonal hybridoma culture comprising a nutrient mediumcontaining a hybridoma that secretes antibody molecules of theappropriate specificity. The culture is maintained under conditions andfor a time period-sufficient for the hybridoma to secrete the antibodymolecules into the medium. The antibody-containing medium is thencollected. The antibody molecules can then be further isolated by wellknown techniques.

Media useful for the preparation of these compositions are both wellknown in the art and commercially available and include syntheticculture media, inbred mice and the like. An exemplary synthetic mediumis Dulbecco's minimal essential medium (DMEM; Dulbecco et al., Virol.8:396, 1959) supplemented with 4.5 g/L glucose, 20 nM glutamine, and 20%fetal calf serum. An exemplary inbred mouse strain is the Balb/c.

Other methods of producing a monoclonal antibody, a hybridoma cell, or ahybridoma cell culture also are well known. See, for example, the methodof isolating monoclonal antibodies from an immunological repertoire asdescribed by Sastry et al. (1989) Proc. Natl. Acad. Sci. USA,86:5728-5732; and Huse et al. (1989) Science, 246:1275-1281.

Also contemplated by this invention is the hybridoma cell, and culturescontaining hybridoma cells that produce monoclonal antibodies of thisinvention. Particularly preferred is a hybridoma cell line that secretesmonoclonal antibody mAb HUI77, mAb HUIV26, or mAb XL313.

The invention contemplates, in one embodiment, a monoclonal antibodythat has the immunoreaction characteristics of Mab HUI77, Mab HUIV26, orMab XL313.

It also is possible to determine, without undue experimentation, if amonoclonal antibody has an equivalent specificity (immunoreactioncharacteristics) as a monoclonal antibody of this invention byascertaining whether the former prevents the latter from binding to apreselected target molecule. If the monoclonal antibody being testedcompetes with the monoclonal antibody of the invention, as shown by adecrease in binding by the monoclonal antibody of the invention instandard competition assays for binding to the target molecule whenpresent in the solid phase, then it is likely that the two monoclonalantibodies bind to the same, or a closely related, epitope.

An additional way to determine whether a monoclonal antibody has thespecificity of a monoclonal antibody of the invention is to determinethe amino acid residue sequence of the CDR regions of the antibodies inquestion. Antibody molecules having identical, or functionallyequivalent, amino acid residue sequences in their CDR regions have thesame binding specificity. Methods for sequencing polypeptides are wellknown in the art. This does not suggest that antibodies with distinctCDR regions cannot bind to the same epitope.

The immunospecificity of an antibody, its target molecule bindingcapacity, and the attendant affinity the antibody exhibits for theepitope, are defined by the epitope with which the antibodyimmunoreacts. The epitope specificity is defined at least in part by theamino acid residue sequence of the variable region of the heavy chain ofthe immunoglobulin the antibody, and in part by the light chain variableregion amino acid residue sequence.

Use of the term “having the binding specificity of” indicates thatequivalent monoclonal antibodies exhibit the same or similarimmunoreaction (binding) characteristics and compete for binding to apreselected target epitope.

Humanized monoclonal antibodies offer particular advantages over murinemonoclonal antibodies, particularly insofar as they can be usedtherapeutically in humans. Specifically, human antibodies are notcleared from the circulation as rapidly as “foreign” antigens, and donot activate the immune system in the same manner as foreign antigensand foreign antibodies. Methods of preparing “humanized” antibodies aregenerally well known in the art, and can readily be applied to theantibodies of the present invention.

Thus, the invention contemplates, in one embodiment, a monoclonalantibody of this invention that is humanized by grafting to introducecomponents of the human immune system without substantially interferingwith the ability of the antibody to bind antigen.

The antibody of the invention can also be a fully human antibody such asthose generated, for example, by selection from an antibody phagedisplay library displaying human single chain or double chain antibodiessuch as those described in de Haard, H. J. et al. (1999) J. Biol. Chem.274:18218-30 and in Winter, G. et al. (1994) Annu. Rev. Immunol.12:433-55.

Polypeptides

Antagonists of denatured collagen also can be polypeptides or peptides.The term polypeptide refers to a sequence of 3 or more amino acidsconnected to one another by peptide bonds between the alpha-amino groupand carboxy group of contiguous amino acid residues. The term peptide asused herein refers to a linear series of two or more connected to one tothe other as in a polypeptide.

In one embodiment, the invention contemplates denatured collagenantagonists in the form of polypeptides. A polypeptide antagonist ofdenatured collagen can be any peptide or polypeptide capable of bindingto a denatured collagen, but binds to the native form of the collagenwith substantially reduced affinity.

The identification of preferred denatured collagen antagonist peptideshaving selectivity for denatured collagen can readily be identified in atypical inhibition of binding assay, such as the ELISA assay describedin the Examples.

Peptide and polypeptide antagonists can be generated by a number oftechniques known to one of skill in the art. For example, a two hybridsystem (e.g., Fields, S. (1989) Nature 340:245-6) can use a fragment ofa collagen as “bait” for selecting protein antagonists from a librarythat bind to the collagen peptide. The library of potential antagonistscan be derived from a cDNA library, for example. In another embodiment,the potential antagonists can be variants of known collagen bindingproteins. Such proteins can be randomly mutagenized or subjected to geneshuffling, or other available techniques for generating sequencediversity.

Peptide and polypeptide antagonists of the invention also can begenerated by techniques of molecular evolution. Libraries of proteinscan be generated by mutagenesis, gene shuffling or other availabletechniques for generating molecular diversity. Protein poolsrepresenting numerous variants can be selected for their ability to bindto denatured collagen, for instance by passing such protein pools over asolid matrix to which a denatured collagen has been attached. Elutionwith gradients of salt, for example, can provide purification ofvariants with affinity for the denatured collagen. A negative selectionstep also can be included whereby such pools are passed over a solidmatrix to which native collagens have been attached. The filtrate willcontain those variants within the pool that have a reduced affinity forthe native form of the collagen.

Peptide and polypeptide antagonists of the invention also can begenerated by phage display. A randomized peptide or protein can beexpressed on the surface of a phagemid particle as a fusion with a phagecoat protein. Techniques of monovalent phage display are widelyavailable (see, e.g., Lowman H. B. et al. (1991) Biochemistry30:10832-8.) Phage expressing randomized peptide or protein librariescan be panned with a solid matrix to which a native collagen moleculehas been attached. Remaining phage do not bind native collagens, or bindnative collagens with substantially reduced affinity. The phage are thenpanned against a solid matrix to which a denatured collagen has beenattached. Bound phage are isolated and separated from the solid matrixby either a change in solution conditions or, for a suitably designedconstruct, by proteolytic cleavage of a linker region connecting thephage coat protein with the randomized peptide or protein library. Theisolated phage can be sequenced to determine the identity of theselected antagonist.

In another embodiment, a polypeptide includes any analog, fragment orchemical derivative of a polypeptide whose amino acid residue sequenceis shown herein so long as the polypeptide is an antagonist of denaturedcollagen, but not of native collagen. Therefore, a present polypeptidecan be subject to various changes, substitutions, insertions, anddeletions where such changes provide for certain advantages in its use.In this regard, a denatured collagen antagonist polypeptide of thisinvention corresponds to, rather than is identical to, the sequence of arecited peptide where one or more changes are made and it retains theability to function as a denatured collagen antagonist in one or more ofthe assays as defined herein.

Thus, a polypeptide can be in any of a variety of forms of peptidederivatives, that include amides, conjugates with proteins, cyclizedpeptides, polymerized peptides, analogs, fragments, chemically modifiedpeptides, and like derivatives.

Other Antagonists

Antagonists of the invention also can be small organic molecules, suchas those natural products, or those compounds synthesized byconventional organic synthesis or combinatorial organic synthesis.Compounds can be tested for their ability to bind to a denaturedcollagen for example by using the column binding technique describedabove. Compounds also are selected for reduced affinity for the nativeform of the collagen by a similar column binding technique.

Antagonists of the invention also can be non-peptidic compounds.Suitable non-peptidic compounds include, for example, oligonucleotides.Oligonucleotides as used herein refers to any heteropolymeric materialcontaining purine, pyrimidine and other aromatic bases. DNA and RNAoligonucleotides are suitable for use with the invention, as areoligonucleotides with sugar (e.g., 2′ alkylated riboses) and backbonemodifications (e.g., phosphorothioate oligonucleotides).Oligonucleotides may present commonly found purine and pyrimidine basessuch as adenine, thymine, guanine, cytidine and uridine, as well asbases modified within the heterocyclic ring portion (e.g.,7-deazaguanine) or in exocyclic positions. Oligonucleotide alsoencompasses heteropolymers with distinct structures that also presentaromatic bases, including polyamide nucleic acids and the like.

An oligonucleotide antagonist of the invention can be generated by anumber of methods known to one of skill in the art. In one embodiment, apool of oligonucleotides is generated containing a large number ofsequences. Pools can be generated, for example, by solid phase synthesisusing mixtures of monomers at an elongation step. The pool ofoligonucleotides is sorted by passing a solution containing the poolover a solid matrix to which a denatured collagen or fragment thereofhas been affixed. Sequences within the pool that bind to the denaturedcollagen are retained on the solid matrix. These sequences are elutedwith a solution of different salt concentration or pH. Sequencesselected are subjected to a second selection step. The selected pool ispassed over a second solid matrix to which native collagen has beenaffixed. The column retains those sequences that bind to the nativecollagen, thus enriching the pool for sequences specific for thedenatured collagen. The pool can be amplified and, if necessary,mutagenized and the process repeated until the pool shows thecharacteristics of an antagonist of the invention. Individualantagonists can be identified by sequencing members of theoligonucleotide pool, usually after cloning said sequences into a hostorganism such as E. coli.

Disease Treatment

The present invention relates generally to the discovery that ligationof certain epitopes in denatured collagens but not of native collagensinhibits angiogenesis. This discovery is important because of the rolethat angiogenesis plays in a variety of disease processes. By inhibitingangiogenesis, one can intervene in the disease, ameliorate the symptoms,and in some cases cure the disease.

Where the growth of new blood vessels is the cause of, or contributesto, the pathology associated with a disease, inhibition of angiogenesiswill reduce the deleterious effects of the disease. Examples includepsoriasis, rheumatoid arthritis, diabetic retinopathy, inflammatorydiseases, restenosis, macular degeneration and the like. Where thegrowth of new blood vessels is required to support growth of adeleterious tissue, inhibition of angiogenesis will reduce the bloodsupply to the tissue and thereby contribute to reduction in tissue massbased on blood supply requirements. Examples include growth of tumorswhere neovascularization is a continual requirement in order that thetumor grow beyond a few millimeters in thickness, and for theestablishment of solid tumor metastases.

The methods of the present invention are effective in part because thetherapy is highly selective for angiogenesis and not other biologicalprocesses. As shown in the Examples, only new vessel growth is inhibitedby antagonists of denatured collagens, and therefore the therapeuticmethods do not adversely effect mature vessels. Also as shown in theExamples, an antagonist binds to angiogenic sites in tumors but not tonormal surrounding tissues.

The discovery that ligation of denatured collagens alone can effectivelyinhibit angiogenesis allows for the development of therapeuticcompositions with potentially high specificity, and therefore relativelylow toxicity. Thus although the invention discloses the use ofantibody-based antagonists which have the ability to ligate one or moredenatured collagens, one can design other antagonists that also canspecifically ligate denatured collagens, but not native collagens.

Prior to the discoveries of the present invention, it was not known thatangiogenesis, and any of the processes dependent on angiogenesis, couldbe inhibited in vivo by the use of reagents that antagonize crypticepitopes in collagens, i.e. those that are found in proteolyzed ordenatured collagens, but not in native forms of the same collagens.

Methods for Inhibition of Angiogenesis

The invention provides for a method for the inhibition of angiogenesisin a tissue, and thereby inhibiting events in the tissue which dependupon angiogenesis. Generally, the method comprises administering to thetissue a composition comprising an angiogenesis-inhibiting amount of adenatured collagen antagonist.

As described earlier, angiogenesis includes a variety of processesinvolving neovascularization of a tissue including “sprouting”,vasculogenesis, or vessel enlargement, all of which angiogenesisprocesses involve disruption of extracellular matrix collagen in bloodvessels. With the exception of traumatic wound healing, corpus leuteumformation and embryogenesis, it is believed that the majority ofangiogenesis processes are associated with disease processes andtherefore the use of the present therapeutic methods are selective forthe disease.

There are a variety of diseases in which angiogenesis is believed to beimportant, referred to as angiogenic diseases, including but not limitedto, inflammatory disorders such as immune and non-immune inflammation,chronic articular rheumatism and psoriasis, disorders associated withinappropriate or inopportune invasion of vessels such as diabeticretinopathy, neovascular glaucoma, restenosis, capillary proliferationin atherosclerotic plaques and osteoporosis, and cancer associateddisorders, such as solid tumors, solid tumor metastases, angiofibromas,retrolental fibroplasia, hemangiomas, Kaposi's sarcoma and the likecancers which require neovascularization to support tumor growth. Othersuitable tumors include melanoma, carcinoma, sarcoma, fibrosarcoma,glioma and astrocytoma.

Thus, methods which inhibit angiogenesis in a diseased tissue amelioratesymptoms of the disease and, depending upon the disease, can contributeto cure of the disease. In one embodiment, the invention contemplatesinhibition of angiogenesis, per se, in a tissue. The extent ofangiogenesis in a tissue, and therefore the extent of inhibitionachieved by the present methods, can be evaluated by a variety ofmethods, such as are described in the Examples for detecting proteolyzedor denatured collagen-immunopositive immature and nascent vesselstructures by immunohistochemistry.

As described herein, any of a variety of tissues, or organs comprised oforganized tissues, can support angiogenesis in disease conditionsincluding skin, muscle, gut, connective tissue, joints, bones and thelike tissue in which blood vessels can invade upon angiogenic stimuli.Tissue, as used herein, also encompasses all bodily fluids, secretionsand the like, such as serum, blood, cerebrospinal fluid, plasma, urine,synovial fluid, vitreous humor.

Thus, in one related embodiment, a tissue to be treated is an inflamedtissue and the angiogenesis to be inhibited is inflamed tissueangiogenesis where there is neovascularization of inflamed tissue. Inthis class the method contemplates inhibition of angiogenesis inarthritic tissues, such as in a patient with chronic articularrheumatism, in immune or non-immune inflamed tissues, in psoriatictissue and the like.

The patient treated in the present invention in its many embodiments isdesirably a human patient, although it is to be understood that theprinciples of the invention indicate that the invention is effectivewith respect to all mammals, which are intended to be included in theterm “patient”. In this context, a mammal is understood to include anymammalian species in which treatment of diseases associated withangiogenesis is desirable, particularly agricultural and domesticmammalian species. Such a patient can be, for example, a pig, a cow, ahorse, a goat, a sheep, a mule, a donkey, a dog, a cat, a rabbit, amouse and a rat.

In another related embodiment, a tissue to be treated is a retinaltissue of a patient with diabetic retinopathy, macular degeneration orneovascular glaucoma and the angiogenesis to be inhibited is retinaltissue angiogenesis where there is neovascularization of retinal tissue.

In an additional related embodiment, a tissue to be treated is a tumortissue of a patient with a solid tumor, a metastases, a skin cancer, abreast cancer, a hemangioma or angiofibroma and the like cancer, and theangiogenesis to be inhibited is tumor tissue angiogenesis where there isneovascularization of a tumor tissue. Typical solid tumor tissuestreatable by the present methods include lung, pancreas, breast, colon,laryngeal, ovarian, Kaposi's Sarcoma and the like tissues. Exemplarytumor tissue angiogenesis, and inhibition thereof, is described in theExamples.

Inhibition of tumor tissue angiogenesis is a particularly preferredembodiment because of the important role neovascularization plays intumor growth. In the absence of neovascularization of tumor tissue, thetumor tissue does not obtain the required nutrients, slows in growth,ceases additional growth, regresses and ultimately becomes necroticresulting in killing of the tumor.

Stated in other words, the present invention provides for a method ofinhibiting tumor neovascularization by inhibiting tumor angiogenesisaccording to the present methods. Similarly, the invention provides amethod of inhibiting tumor growth by practicing theangiogenesis-inhibiting methods.

The methods also are particularly effective against the formation ofmetastases because (1) their formation requires vascularization of aprimary tumor so that the metastatic cancer cells can exit the primarytumor and (2) their establishment in a secondary site requiresneovascularization to support growth of the metastases.

In a related embodiment, the invention contemplates the practice of themethod in conjunction with other therapies such as conventionalchemotherapy directed against solid tumors and for control ofestablishment of metastases. The administration of angiogenesisinhibitor is typically conducted during or after chemotherapy, althoughit is preferably to inhibit angiogenesis after a regimen of chemotherapyat times where the tumor tissue will be responding to the toxic assaultby inducing angiogenesis to recover by the provision of a blood supplyand nutrients to the tumor tissue. In addition, it is preferred toadminister the angiogenesis inhibition methods after surgery where solidtumors have been removed as a prophylaxis against metastases.

Insofar as the present methods apply to inhibition of tumorneovascularization, the methods also can apply to inhibition of tumortissue growth, to inhibition of tumor metastases formation, and toregression of established tumors.

Restenosis is a process of smooth muscle cell (SMC) migration aridproliferation at the site of percutaneous transluminal coronaryangioplasty which hampers the success of angioplasty. The migration andproliferation of SMCs associated with blood vessels during restenosis isrelated to the process of angiogenesis which is inhibited by the presentmethods. Therefore, the invention also contemplates inhibition ofrestenosis by inhibiting angiogenic related processes according to thepresent methods in a patient following angioplasty procedures. Forinhibition of restenosis, the denatuted collagen antagonist is typicallyadministered after the angioplasty procedure for from about 2 to about28 days, and more typically for about the first 14 days following theprocedure.

The present method for inhibiting angiogenesis in a tissue, andtherefore for also practicing the methods for treatment ofangiogenesis-related diseases, comprises contacting a tissue in whichangiogenesis is occurring, or is at risk for occurring, with acomposition comprising a therapeutically effective amount of a denaturedcollagen antagonist capable of binding to denatured or proteolyzedcollagen, but not to native forms of the collagen. Thus, the methodcomprises administering to a patient a therapeutically effective amountof a physiologically tolerable composition containing an denaturedcollagen antagonist of the invention.

The dosage ranges for the administration of the denatured collagenantagonist depend upon the form of the antagonist, and its potency, asdescribed further herein, and are amounts large enough to produce thedesired effect in which angiogenesis and the disease symptoms mediatedby angiogenesis are ameliorated. The dosage should not be so large as tocause adverse side effects, such as hyperviscosity syndromes, pulmonaryedema, congestive heart failure, and the like. Generally, the dosagewill vary with the age, condition, sex and extent of the disease in thepatient and can be determined by one of skill in the art. The, dosagealso can be adjusted by the individual physician in the event of anycomplication.

A therapeutically effective amount is an amount of denatured collagenantagonist sufficient to produce a measurable inhibition of angiogenesisin the tissue being treated, i.e., an angiogenesis-inhibiting amount.Inhibition of angiogenesis can be measured in situ byimmunohistochemistry, as described herein, or by other methods known toone skilled in the art.

Potency of a denatured collagen antagonist can be measured by a varietyof means including inhibition of angiogenesis in the CAM assay, in thein vivo rabbit eye assay, in the in vivo chimeric mouse: human assay andthe like assays.

A therapeutically effective amount of a denatured collagen antagonist ofthis invention in the form of a monoclonal antibody is typically anamount such that when administered in a physiologically tolerablecomposition is sufficient to achieve a plasma concentration of fromabout 0.01 microgram (ug) per milliliter (mL) to about 100 ug/mL,preferably from about 1 ug/mL to about 5 ug/mL, and usually about 5ug/mL. Stated differently, the dosage can vary from about 0.1 mg/kg toabout 300 mg/kg, preferably from about 0.2 mg/kg to about 200 mg/kg,most preferably from about 0.5 mg/kg to about 20 mg/kg, in one or moredose administrations daily, for one or several days.

Where the antagonist is in the form of a fragment of a monoclonalantibody, the amount can readily be adjusted based on the mass of thefragment relative to the mass of the whole antibody. A preferred plasmaconcentration in molarity is from about 2 micromolar (uM) to about 5millimolar (mM) and preferably about 100 uM to 1 mM antibody antagonist.

A therapeutically effective amount of a denatured collagen antagonist ofthis invention in the form of a polypeptide, or small molecule, istypically an amount of polypeptide such that when administered in aphysiologically tolerable composition is sufficient to achieve a plasmaconcentration of from about 0.1 microgram (ug) per milliliter (mL) toabout 200 ug/mL, preferably from about 1 ug/mL to about 150 ug/mL. Basedon a polypeptide having a mass of about 500 grams per mole, thepreferred plasma concentration in molarity is from about 2 micromolar(uM) to about 5 millimolar (mM) and preferably about 100 uM to 1 mMpolypeptide antagonist. Stated differently, the dosage per body weightcan vary from about 0.1 mg/kg to about 300 mg/kg, and preferably fromabout 0.2 mg/kg to about 200 mg/kg, in one or more dose administrationsdaily, for one or several days.

The monoclonal antibodies or polypeptides of the invention can beadministered parenterally by injection or by gradual infusion over time.Although the tissue to be treated can typically be accessed in the bodyby systemic administration and therefore most often treated byintravenous administration of therapeutic compositions, other tissuesand delivery means are contemplated where there is a likelihood that thetissue targeted contains the target molecule. Thus, antagonistsincluding monoclonal antibodies, polypeptides, and derivatives thereofcan be administered intravenously, intraperitoneally, intramuscularly,subcutaneously, intracavity, transdermally, topically, intraocularly,orally, intranasally and can be delivered by peristaltic means.

The therapeutic compositions containing a monoclonal antibody or apolypeptide of this invention are conventionally administeredintravenously, as by injection of a unit dose, for example. The term“unit dose” when used in reference to a therapeutic composition of thepresent invention refers to physically discrete units suitable asunitary dosage for the subject, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect in association with the required diluent; i.e.,carrier, or vehicle.

In one preferred embodiment as shown in the Examples, the denaturedcollagen antagonist is administered in a single dosage intravenously.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered and timing depends on the patient to be treated,capacity of the patient's system to utilize the active ingredient, anddegree of therapeutic effect desired. Precise amounts of activeingredient required to be administered depend on the judgement of thepractitioner and are peculiar to each individual. However, suitabledosage ranges for systemic application are disclosed herein and dependon the route of administration. Suitable regimes for administration alsoare variable, but are typified by an initial administration followed byrepeated doses at one or more hour intervals by a subsequent injectionor other administration. Alternatively, continuous intravenous infusionsufficient to maintain concentrations in the blood in the rangesspecified for in vivo therapies are contemplated.

As demonstrated by the present Examples, inhibition of angiogenesis andtumor regression occurs as early as 7 days after the initial contactingwith antagonist. Additional or prolonged exposure to antagonist ispreferable for 7 days to 6 weeks, preferably about 14 to 28 days.

Therapeutic Compositions

The present invention contemplates therapeutic compositions useful forpracticing the therapeutic methods described herein. Therapeuticcompositions of the present invention contain a physiologicallytolerable carrier together with an denature or proteolyzed collagenantagonist as described herein, dissolved or dispersed therein as anactive ingredient. In a preferred embodiment, the therapeutic denaturedcollagen antagonist composition is not immunogenic when administered toa mammal or human patient for therapeutic purposes.

As used herein, the terms “pharmaceutically acceptable”,“physiologically tolerable” and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration to or upon a mammal.

The preparation of a pharmacological composition that contains activeingredients dissolved or dispersed therein is well understood in the artand need not be limited based on formulation. Typically suchcompositions are prepared as injectables either as liquid solutions orsuspensions, however, solid forms suitable for solution, or suspensions,in liquid prior to use also can be prepared. The preparation also can beemulsified.

The active ingredient can be mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredientand in amounts suitable for use in the therapeutic methods describedherein. Suitable excipients are, for example, water, saline, dextrose,glycerol, ethanol or the like and combinations thereof. In addition, ifdesired, the composition can contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agentsand the like which enhance the effectiveness of the active ingredient.

The therapeutic composition of the present invention can includepharmaceutically acceptable salts of the components therein.Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide) that are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, tartaric, mandelic and the like.Salts formed with the free carboxyl groups also can be derived frominorganic bases such as, for example, sodium, potassium, ammonium,calcium or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.Particularly preferred are the salts of TFA and HCl.

Physiologically tolerable carriers are well known in the art. Exemplaryof liquid carriers are sterile aqueous solutions that contain nomaterials in addition to the active ingredients and water, or contain abuffer such as sodium phosphate at physiological pH value, physiologicalsaline or both, such as phosphate-buffered saline. Still further,aqueous carriers can contain more than one buffer salt, as well as saltssuch as sodium and potassium chlorides, dextrose, polyethylene glycoland other solutes.

Liquid compositions also can contain liquid phases in addition to and tothe exclusion of water. Exemplary of such additional liquid phases areglycerin, vegetable oils such as cottonseed oil, and water-oilemulsions.

A therapeutic composition contains an angiogenesis-inhibiting amount ofan denatured collagen antagonist of the present invention, typicallyformulated to contain an amount of at least 0.1 weight percent ofantagonist per weight of total therapeutic composition. A weight percentis a ratio by weight of inhibitor to total composition. Thus, forexample, 0.1 weight percent is 0.1 grams of inhibitor per 100 grams oftotal composition.

An antibody can be conjugated with cytotoxins, cytotoxic agents, frodelivery to a to tumor or other tissue undergoing angiogenesis. Suchconjugates can be made with a cytolysin or an exotoxin, for examplericin A, diphtheria toxin A, or Pseudomonas exotoxin and fragmentsthereof. The cytotoxic agent can also be a radioactively labeled with anisotope so as to locally deliver a toxic dose of radioactivity to anangiogenic tissue.

Antagonists of the invention can also be used to deliver an enzyme to atarget wherein the enzyme is capable of converting a prodrug into anactive form of the drug. antibody-directed enzyme activated prodrugtherapy (ADEPT) (see, e.g., Syrigos, K. N. (1999) Anticancer Res.19:605-13). Briefly, an antagonist of the invention is conjugated withan enzyme, such as a lactamase, protease or esterase, that can convert anon-toxic or inactive prodrug into a toxic or active drug. Because theantagonist of the invention localizes to sites of angiogenesis, andparticularly to sites of tumors or metastases, toxic drugs can bedirected to the

Detection Methods

Antagonists of the invention also are suitable for detection ofangiogenesis in tissues.

For example, where the antagonist is an antibody, the antagonist can beused in immunohistochemical techniques to stain tissues ex vivo.Immunological techniques such as immunostaining and ELISA are describedin, for example, Receptor Binding Techniques, Methods in MolecularBiology. 106. ed. M. Keen. Humana Press, 1999; Brooks et al. (1998) Cell92:391-400; Brooks et al. (1996) Cell 85:683-693; and Brooks et al.(1993) J. Cell. Biol. 122:1351-1359.

The antagonist of the invention, once bound to the target tissue can bedetected either directly or indirectly. Direct detection can bepreformed on antagonists that comprise a detectable label such as afluorochrome, a radioactive tag, paramagnetic heavy metal or diagnosticdye.

Alternatively, detection can occur through a secondary interaction. Forexample, a detectably labeled antibody that recognizes the antagonistcan be used to visualize the location of the antagonist. For example, ifthe antagonist is a monoclonal antibody of mouse origin, a goatanti-mouse antibody that is suitably labeled can be used. The Examplesdescribe the use, for example, of a goat anti-mouse peroxidaseconjugated antibody. One of skill in the art can determine suitablesecondary antibodies for use with various antagonists.

For in vivo detection, it is preferable to use a detectably labeledantagonist. The labeled antagonist is administered to a patientintravenously, intramuscularly, etc . . . Labels suitable for detectionwithin a patient are particularly preferred. For example,paramagnetically labeled can be detected by magnetic resonance imaging.Radioactively tagged antagonists also can be detected.

EXAMPLES Example 1

Monoclonal Antibody HUI77

This example describes the generation of a denatured collagen specificmonoclonal antibody, Mab HUI77.

Mab HUI77 was generated and isolated by the immunological techniquetermed subtractive immunization (S.I). The subtractive immunizationtechnique allows one to experimentally manipulate the immune responsewithin mice to selectively enhance an immune response to a rare and/orlow abundant epitope within a mixture of common highly antigenicepitopes. Briefly, female BALB/c mice were injected intraperitoneallywith either native human triple helical collagen type-I or type-IV. At24 and 48 hours following the injections of triple helical collagen, themice were injected with the tolerizing agent cyclophosphamide to killactivated B-cells that would produce antibodies directed to commonimmunodorninant epitopes within native triple helical collagen type-Iand type-IV. Following the tolerization protocol, the mice were nextinjected with thermally denatured human collagen type-I or type-IV tostimulate an immune response to epitopes exposed following thermaldenaturation. Collagen was denatured by boiling for 15 minutes. Theinjections of thermally denatured collagen type-I and type-IV were givenevery three weeks for a total of 4 to 5 injections. Sera from each mousewas tested for immunoreactivity with both native triple helical anddenatured collagens. The mice demonstrating the highest titer forreactivity to denatured collagen as compared to triple helical collagenwere used for the production of hybridomas. Spleen cells from theselected mice were fused with myeloma cells by standard techniques.Individual hybridoma clones were tested for the production of antibodyto either triple helical or denatured collagen type-I and type-IV.Hybridoma clones were selected that produced antibodies thatdemonstrated a selective reactivity to denatured collagen type-I ortype-IV as compared to native triple helical collagens type-I andtype-IV. Mabs were purified by standard techniques.

As shown in FIG. 1, HUI77 was shown to specifically recognize denaturedcollagens type-I and type-IV but binds to native triple helicalcollagens type-I and type-IV with substantially reduced affinity. Inparticular, HUI77 binds to denatured collagen type-I with an apparentreactivity of at least about 10-fold higher than that of native collagentype-I as measured by ELISA. HUI77 also binds to denatured collagentype-IV with an affinity of about 10-fold higher than for nativecollagen type-IV. In addition, Mab HUI77 does not bind substantially to,other matrix components such as laminin, fibronectin, vitronectin orfibrinogen, thus demonstrating its specificity to a cryptic epitopewithin collagens type-I and type-IV.

Mab HUI77 also is specific for other denatured collagens and binds thenative forms of these collagens with substantially reduced affinity. Asshown in FIG. 2, HUI77 also binds denatured collagens III, IV and V withabout 7-fold, about 8-fold, and about 10-fold more tightly than therespective native forms of these collagens using ELISA.

Example 2

Detection of Solid Tumors

This example shows that antagonists of the invention can be used todetect denatured collagens in tumorous tissue. Monoclonal antibodyHUI77, described in Example 1, was used to indirectly immunostain normaland tumorous tissue. As shown in FIG. 3, indirect immunofluoresenceanalysis using Mab HUI77 of human melanoma tumor biopsies as well as ofM21 melanoma tumor grown in full thickness human skin indicate thegeneration of denatured forms of collagens associated with humanmelanoma tumors in vivo. Importantly, little if any evidence ofdenatured collagen was detected in normal tissues in the absence oftumors, suggesting that denatured collagen may be a specific marker ofsolid human tumors.

Example 3

Detection of Angiogenesis in Human Tumors

This example demonstrates that antagonists of the invention can be usedto detect angiogenesis in human tumors.

Indirect immunofluoresence analysis, shown in FIG. 4, of human melanomatumor biopsies demonstrates the generation and localization of denaturedcollagen surrounding human tumor associated blood vessels in vivo.Importantly, little if any evidence of denatured collagen was detectedwith Mab HUI77 surrounding normal blood vessels in the absence oftumors, suggesting that denatured collagen may be a specific marker ofangiogenic tumor associated blood vessels.

Example 4

Antagonists Inhibit Endothelial Cell Adhesion and Migration

This example demonstrates that certain antagonists of the invention caninhibit human endothelial cell adhesion to denatured collagens.

Mab HUI77 showed the capacity to inhibit human endothelial cell adhesionto denatured collagen type-I by approximately 40% as compared to controlantibody. These findings, summarized in FIG. 5, suggest that Mab HUI77binds to a cryptic epitope within collagen type-I that is at leastpartially involved in endothelial cell adhesion to denatured collagen-I.Since endothelial cell adhesive processes are thought to play a role intumor growth and angiogenesis, this function blocking antibody may havean effect on angiogenesis and tumor growth in vivo.

Mab HUI77 also showed the capacity to inhibit human endothelial cellmigration on denatured collagen-I by approximately 80% as compared toeither control antibody or no treatment, as shown in FIG. 6. Thesefindings suggest that Mab HUI77 binds to a cryptic epitope withincollagen type-I that plays a significant role in cellular migration ondenatured collagen-I. Given that cell migration is thought to play aimportant role in tumor metastasis and angiogenesis, and that denaturedcollagen was detected in association with malignant tumor cells andangiogenic blood vessels, this function blocking antibody may have asignificant impact on angiogenesis and tumor growth and metastasis invivo.

Example 6

Inhibition of Angiogenesis by Monoclonal Antibody HUI77

This example shows that antagonists of the invention effectively inhibitangiogenesis in the chick CAM assay.

Furthermore, systemic administration of Mab HUI77 inhibited βFGF inducedangiogenesis by approximately 90% as compared to controls (FIGS. 7 and8). Angiogenic index was measured by counting the number of blood vesselbranch points in the chick CAM assay (FIG. 8). Importantly, no toxicside effects were noted in the embryos during the assay period.Moreover, few if any effects from this Mab were noted on normalquiescent blood vessels. It is possible that much lower concentration ofMab might be used and result in similar effects. These findings indicatethat Mab HUI77 is a potent anti-angiogenic reagent that may havesignificant clinical applications.

Example 7

Inhibition of Tumor Growth by Mab HUI77

This example shows that antagonists of the invention effectively inhibittumor growth in melanoma tumors in vivo.

Systemic administration of Mab HUI77 inhibited Melanoma tumor growth byapproximately 53% as compared to controls, as shown in FIGS. 9 and 10.Importantly, no toxic side effects were noted in the embryos during theassay period. Moreover, little if any effects from this Mab were notedon adjacent tissue. It is possible that much lower concentration of Mabmight be used and result in similar effects. These findings indicatethat Mab HUI77 is a potent anti-tumor reagent that may have significantclinical applications.

Example 8

Monoclonal Antibody HUIV26

Mab HUIV26 was generated by the immunological technique termedsubtractive immunization (S.I.) as outlined in Example 1. As shown inFIG. 11, HUIV26 was shown to specifically recognize denatured collagentype-IV but does not bind to native triple helical collagen type-I ortype-IV. In addition, Mab HUIV26 does not bind to other matrixcomponents such as Laminin, Fibronectin, Vitronectin or fibrinogen, thusdemonstrating its specificity to a cryptic epitope within collagentype-IV.

As shown in FIG. 11B, the cryptic site(s) in type-IV collagen recognizedby Mab HUIV26 are revealed after exposure to HUVEC conditioned media.The amount of reactivity with Mab HUIV26 increased over the 24 hourperiod examined. This indicates that these endothelial cells secrete aprotease capable of unmasking the cryptic site in type-IV collagenrecognized by Mab HUIV26. The protease produced by HUVEC that unmasksthe cryptic site in type-IV collagen is inhibited by the chelator, EDTA(FIG. 11B). This suggests that a metalloprotease is responsible forinitiating the unmasking of the cryptic site(s) in type-IV collagen. Asshown in FIG. 12, the metalloprotease, MMP-2, colocalizes with thecryptic site(s) in type-IV collagen reacting with Man HUIV26 inangiogenic sites in CAM tissue. The serine protease inhibitor,aprotinin, had little effect on unmasking of cryptic site(s) in type-IVcollagen after one hour of incubation with HUVEC conditioned media (FIG.11B). At the 6 and 24 hour time points, the presence of aprotininblocked 40 and 70 percent respectively of the reactivity observed in theabsence of the protease inhibitor. This suggests that at the later timepoints (6 and 24 hour),serine proteases contribute to the unmasking ofcryptic site(s) in type-IV collagen.

Example 9

Detection of Angiogenesis and Tumors with HUIV26

This example shows that an antagonist can be used to detect angiogenicprocesses within tissues.

As shown in FIG. 12, indirect immunofluoresence analysis of chick CAMtissue indicates the generation and localization of denatured collagentype-IV associated with either βFGF or tumor induced angiogenic bloodvessels in vivo. Importantly, little if any evidence of denaturedcollagen-IV was detected in normal CAM tissues in the absence of bFGF ortumors, suggesting that denatured collagen-IV may be a specific markerof angiogenic blood vessels in vivo.

Indirect immunofluoresence analysis, shown in FIG. 13, of human melanomatumor biopsies demonstrates the generation and localization of denaturedcollagen-IV surrounding human tumor associated blood vessels in vivo.Importantly, little if any evidence of denatured collagen-IV wasdetected surrounding normal blood vessels in the absence of tumors,suggesting that denatured collagen may be a specific marker ofangiogenic tumor associated blood vessels.

Example 10

Inhibition of Cell Migration and Adhesion by Mab HUIV26

This example shows that an Mab antagonist can inhibit endothelial cellmigration and adhesion.

Mab HUIV26 showed the capacity to inhibit human endothelial celladhesion to denatured collagen-IV by approximately 70% as compared tocontrol antibody (FIG. 14). These findings suggest that Mab HUIV26 bindsto a cryptic epitope within collagen type-IV that is at least partiallyinvolved in endothelial cell adhesion to denatured collagen-IV. Giventhe tissue distribution of collagen type-IV and the fact that cellularadhesive processes are thought to play a role in tumor growth andangiogenesis, this function blocking antibody may have an significanteffect on angiogenesis and tumor growth in vivo.

Mab HUIV26 showed the capacity to inhibit human endothelial cellmigration on denatured collagen-IV by approximately 70% as compared tocontrol antibody or no treatment. These findings suggest that Mab HUIV26binds to a cryptic epitope within collagen type-IV that plays asignificant role in cellular migration on denatured collagen-IV. Giventhat cell migration is thought to play a important role in tumormetastasis and angiogenesis, and that denatured collagen was detected inassociation with angiogenic blood vessels, this function blockingantibody may have a significant impact on angiogenesis, tumor growth andmetastasis in vivo.

Example 11

Inhibition of Angiogenesis by Systemic Administration of HUIV26

Systemic administration of Mab HUIV26 inhibited bFGF inducedangiogenesis by approximately 90% as compared to controls, as shown inFIGS. 16 and 17. Importantly, no toxic side effects were noted in theembryos during the assay period. Moreover, few, if any, effects fromthis Mab were noted on normal quiescent blood vessels. It is possiblethat much lower concentration of Mab might be used and result in similareffects. These findings indicate that Mab HUIV26 is a potentanti-angiogenic reagent that may have significant clinical applications.

Example 12

Inhibition of Tumor Growth

Systemic administration of Mab HUIV26 inhibited melanoma tumor growth byapproximately 80% as compared to controls (FIG. 18). Importantly, notoxic side effects were noted in the embryos during the assay period.Moreover, little if any effects from this Mab were noted on adjacenttissue. Additional experiments are now under way to determine an IC50value since it is possible that much lower concentration of Mab might beused and result in similar effects. These findings indicate that MabHUIV26 is a potent anti-tumor reagent that may have significant clinicalapplications.

Systemic administration of Mab HUIV26 inhibited melanoma tumor growth inSCID mice. After 24 days of treatment, Mab HUIV26 treated mice had anaverage tumor volume less than 5 percent that observed in mice treatedwith a control Mab or in mice not receiving treatment.

Example 13

Epitope Specificity of HUIV26

This example shows that HUIV26 is not binding to RGD sequences withindenatured collagens.

As shown in FIG. 22, Mab HUIV26 fails to react with any of theimmobilized RGD (arginine-glycine-aspartic acid) containing peptidesfound in type-IV collagen (Table 1). Six different soluble RGDcontaining peptides found in collagens failed to block the recognitionof immobilized denatured type-IV collagen by Mab HUIV26 (FIG. 22). Thesedata suggest that Mab HUIV26 does not recognize RGD sequences found intype-IV collagen. TABLE 1 RGD Domains of Human Collagen Type-IV SEQ IDAmino Acid Position in NO Sequence Human Collagen Type-IV 1C-PGSRGDTGP-C α1 (IV) Chain (594-602) 2 C-SGPRGDPGL-C α1 (IV) Chain(914-922) 3 C-KGSRGDPGT-C α1 (IV) Chain (965-973) 4 C-KGARGDPGF-C α2(IV) Chain (359-367) 5 C-PGPRGDAGV-C α2 (IV) Chain (781-789) 6C-PGDRGDPGD-C α2 (IV) Chain (865-873) 7 C-SGDRGDAGF-C α2 (IV) Chain(886-894) 8 C-KGSRGDPGP-C α2 (IV) Chain (967-975) 9 C-IGSRGDKGA-C α2(IV) Chain (1066-1074) 10 C-PGERGDPGE-C α2 (IV) Chain (1245-1253) 11C-PGFRGDEGP-C α2 (IV) Chain (1489-1497)

Example 14

Monoclonal Antibody XL313

Mab XL313 was generated by immunization with a synthetic peptide, thesequence of which was derived from human collagen type-I. The sequencewas chosen because it is buried within the three dimensional structureof collagen type-I. The sequence of the 11 amino acid residue syntheticpeptide used was: SEQ ID NO 12: CysGlnGlyProArgGlyAspLysGlyGluCys

The KGE (LysGlyGlu) sequence was found to be very important for XL313recognition. Mab XL313 specifically binds to peptides of SEQ ID NO 1,but has substantially decreased binding affinity for peptides in whichthe KGE sequence has been mutated: SEQ ID NO 13:CysGlnGlyProArgGlyAspAlaAlaAlaCys

Mab termed XL313 is a highly specific antibody that reacts withproteolyed/denatured collagen type-I. Importantly, XL313 does not reactwith the native triple helical form of collagen type-I. As shown in FIG.27, Mab XL313 recognizes a cryptic domain of human collagen-I, but notother similar peptides. These data suggest that Mab XL313 may be auseful reagent to assess the role of the cryptic collagen domain definedby human collagen peptide-2 in angiogenesis and tumor growth. As shownin FIG. 28, Mab XL313 specifically recognizes a cryptic domain withinhuman collagen-I that is not exposed within the mature triple helicalconformation. Moreover, this cryptic domain appears to be specific tocollagen-I as XL313 does not cross react with native triple helicalcollagen-IV.

Example 15

Mab XL313 Inhibits Cell Adhesion and Migration

As shown in FIG. 23, at the end of 5 hours, incubation HUVECs allowed toattach to native collagen-I formed a confluent monolayer. In contrast,HUVECs attached to denatured collagen began to migrate andmorphologically reorganized to form cord-like structures. These datasuggest that cryptic domains hidden within the three dimensionalstructure of human collagen-I, that are not assessable in its nativetriple helical state may play a role in endothelial morphogenesis andcord formation.

Example 16

Denatured Collagen Suppresses Apoptosis

As shown in FIG. 24, Human endothelial cell interactions withproteolyzed collagen suppresses apoptosis as compared to either nativecollagen-I or endothelial cells kept in suspension. These data suggestthat cryptic domains hidden within the three dimensional structure ofhuman collagen-I, that are not accessable in its native triple helicalstate may play a role in endothelial cell survival.

Example 17

The Epitope Recognized by XL313

As shown in FIG. 25, human collagen cryptic peptide-2 appears to supportendothelial cell survival and cord formation, while similar crypticpeptides present within human collagen-I show little if any effect.These data suggest that the cryptic region of collagen-I defined bypeptide-2 may play an important role in angiogenesis and tumor growth invivo.

Example 18

Role of Integrins

As shown in FIG. 26, integrin αvβ3 appears to play a major role inmediating cellular interactions with all the cryptic peptide domains ofcollagen-I tested. Interestingly, peptide-2 was also dependent on β1integrin interaction. These data suggest that peptide-2 supportscellular interactions by 2 distinct integrins.

Example 19

Mab XL313 Inhibits Angiogenesis and Tumor Growth

As shown in FIGS. 29 and 30, Systemic administration of Mab XL313inhibited angiogenesis in the Chick CAM model by over 95% as compared tocontrols. These data suggest that the cryptic domain of collagen-Idefined by Mab XL313 plays an important role in angiogenesis.

As shown in FIG. 31, Mab XL313 potentially inhibits HT1080 fibrosarcomatumor growth in vivo. These findings indicate that the cryptic domaindefined by Mab XL313 may play a significant role in regulating tumorgrowth in vivo.

Example 20

Proteolysis of Collagen is Important for Tumor Growth

As shown in FIG. 32, transgenic mice in which a mutation was introducedwithin the MMP cleavage site of collagen-I molecule was used in thisexperiment since these mice have impaired ability to proteolyze theircollagen-I. Importantly, B16 melanoma cells exhibited little, if any,ability to form tumors in mice that exhibit impaired collagen-Iproteolysis as compared to wild-type control mice. These data suggestthat proteolysis of collagen-I plays an important role in tumor growthin vivo.

As was demonstrated with B16 melanoma cells, Lewis lung carcinoma cellsalso exhibited little, if any, capacity to form tumors when injected inCol a1 B6 transgenic mice which have impaired ability to proteolyzetheir collagen-I, while Lewis lung carcinoma cells form large rapidlygrowing tumors in control B6 mice (FIG. 33). Importantly, Mab XL313,specifically directed to a cryptic domain within collagen-I which isonly exposed following proteolysis, inhibited Lewis lung carcinoma tumorgrowth in wild-type B6 mice by approximately 80%. The findings suggestthat proteolytic exposure of the cryptic domain of collagen-I in vivomay play an important role in tumor growth. Moreover, these data suggestthat Mab XL313 is a specific inhibitor of angiogenesis and tumor growthin vivo.

All of the following publications which are cited in the body of theinstant specification are hereby incorporated by reference in theirentirety.

It is also to be appreciated that the foregoing description of theinvention has been presented for purposes of illustration andexplanation and is not intended to limit the invention to the precisemanner of practice herein. It is to be appreciated therefore, thatchanges may be made by those skilled in the art without departing fromthe spirit of the invention and that the scope of the invention shouldbe interpreted with respect to the following claims.

1. An antagonist that specifically binds to a denatured collagen orcollagens but binds to the native triple helical form of each of saidcollagens with substantially reduced affinity and wherein saidantagonist inhibits angiogenesis, wherein the antagonist is an antibody.2. The antagonist of claim 1 wherein said reduced affinity is about3-fold lower than that for said denatured collagen.
 3. The antagonist ofclaim 1 wherein said reduced affinity is about 5-fold lower than thatfor said denatured collagen.
 4. The antagonist of claim 1 wherein saidreduced affinity is about 10-fold lower than that for said denaturedcollagen.
 5. The antagonist of claim 1 wherein said denatured collagenis denatured collagen type-I, denatured collagen type-II, denaturedcollagen type-III, denatured collagen type-IV, denatured collagentype-V, or any combination thereof.
 6. The antagonist of claim 5 whereinsaid denatured collagen is denatured collagen type-I.
 7. The antagonistof claim 5 wherein said denatured collagens are denatured collagentype-I and denatured collagen type-IV.
 8. The antagonist of claim 5wherein said denatured collagens are denatured collagen type-II,denatured collagen type-III, and denatured collagen type-V.
 9. Theantagonist of claim 1 wherein said antagonist is a monoclonal antibodyor a fragment thereof.
 10. The antagonist of claim 1 wherein saidantagonist is a polyclonal antibody.
 11. The antagonist of claim 1wherein said antagonist is a humanized or chemically modified monoclonalantibody.
 12. The antagonist of claim 1 wherein said antagonist isconjugated to cytotoxic or cytostatic agents.
 13. A method forinhibiting angiogenesis in a tissue comprising administering atherapeutically-effective amount of the antagonist of claim
 1. 14. Themethod of claim 13 wherein said antagonist is administered inconjunction with chemotherapy or radiation.
 15. The method of claim 13wherein the tissue is inflamed and angiogenesis is occurring.
 16. Themethod of claim 15 wherein the tissue is present in a mammal.
 17. Themethod of claim 15 wherein the tissue is arthritic, ocular, retinal, ahemangioma, an angiofibroma, a Kaposi's sarcoma, or a solid tumor of thelung, pancreas, breast, colon, larynx, ovary or skin.
 18. A method forinhibiting tumor growth or metastasis comprising administering atherapeutically-effective amount of the antagonist of claim
 1. 19. Themethod of claim 18 wherein said antagonist is administeredintravenously, transdermally, intrasynovially, intramuscularly,intratumorally, intraocularly, intranasally, topically or orally. 20.The method of claim 18 wherein said antagonist is administered inconjunction with chemotherapy or radiation.
 21. The method of claim 18wherein the tumor or metastasis is a melanoma, carcinoma, sarcoma,fibrosarcoma, glioma, astrocytoma, angiofibroma, a solid tumor of thelung, pancreas, breast, colon, larynx, ovary or skin, or Kaposi'ssarcoma.
 22. A method of inhibiting psoriasis, macular degeneration, orrestenosis in a tissue comprising administering atherapeutically-effective amount of the antagonist of claim
 1. 23. Themethod of claim 22 wherein said antagonist is administeredintravenously, transdermally, intrasynovially, intramuscularly,intratumorally, intraocularly, intranasally, topically or orally. 24.The method of claim 22 wherein said antagonist is administered inconjunction with chemotherapy or radiation.
 25. A method of detectingangiogenesis in a tissue by exposing said tissue to the antagonist ofclaim
 1. 26. The method of claim 25 wherein said tissue is ex vivo. 27.The method of claim 25 wherein said tissue is in vivo and antagonist isadministered intravenously, transdermally, intrasynovially,intramuscularly, intratumorally, intraocularly, intranasally, topicallyor orally.
 28. The method of claim 25 wherein said antagonist isconjugated to a fluorochrome, radioactive tag, paramagnetic heavy metal,diagnostic dye or enzyme.
 29. A method of detecting tumors or tumorinvasion in a tissue by exposing said tissue to the antagonist ofclaim
 1. 30. The method of claim 29 wherein said tissue is ex vivo. 31.The method of claim 29 wherein said tissue is in vivo and antagonist isadministered intravenously, transdermally, intrasynovially,intramuscularly, intratumorally, intraocularly, intranasally, topicallyor orally.
 32. The method of claim 29 wherein said antagonist isconjugated to a fluorochrome, radioactive tag, paramagnetic heavy metalor diagnostic dye.